215 In vitro evaluation of reprogramming at 20% or 5% oxygen tension in adult equine fibroblasts

Abstract

The reprogramming process suffers from effects of external and internal factors, one of which is oxygen concentration, because it plays important roles in several processes of the cell. The objective of this study was to evaluate the effect of oxygen during reprogramming of equine adult fibroblasts. Fibroblasts were isolated from the skin of one male and immediately cultured under high (20%, HO group) or low (5%, LO group) oxygen tension. Generation of induced pluripotent stem cells (iPSCs) was performed by transduction with human sequences of OCT4, SOX2, KLF4, and c-MYC cDNAs. Morphology evaluation and RNA samples collection were performed at the day of transduction (Day 0), and then every 3 days (Days 3, 6, 9, 12, 15, and 18). Six days after transduction, cells were passed to mouse embryonic fibroblast (MEFs). A third group, in which the cells were cultured in high oxygen and passed to low oxygen, was formed (high to low group, HLO) and analysed. The equine (e)iPS colonies were evaluated in terms of reprogramming efficiency, morphology, detection of alkaline phosphatase, and qRT-PCR for markers of pluripotency (OCT4, REX1, NANOG, and SOX2 genes), glycolysis (PFKM and GAPDH), mitochondrial fission and fusion (DNM1L and MFN1), hypoxia (HIF1α, HIF2α, and VEGFA), and presence of the exogenous vector (HS). The values of the target genes were normalized by the average values of the housekeeping genes (HPRT1 and PPIA) and the fold changes were calculated using the 2^(−ΔCT) equation. After 10, 11 and 14 days of transduction, the first colonies appeared in the LO, HO, and HLO groups. Their reprogramming efficiency was 1.67%, 0.08%, and 0.025%, respectively. The cells were primarily identified by their morphology: colonies with well-defined edges and cells with a high nuclear/cytoplasm ratio. Our results indicated that genes related to pluripotency increased at first, and then some decreased at Day 18, probably due to the time of picking the first colonies. The LO group showed more elevated levels of these genes than HO group, in accordance with the higher reprogramming efficiency found in LO. Lentiviral vector expression peaked at Day 3 and then decreased at Day 3 in LO and at Day 12 in HO. Glycolysis genes were less expressed in LO, which may suggest a successful metabolism change. The mitochondrial gene MF1 was higher in HO than in LO, and expression of DNM1L increased at Day 12 in the LO group. Expression of VEGFA, a direct target of HIF1α, behaved similarly. Expression of HIF1α and HIF2α differed from expected, being higher in LO than in HO. The expression of HIF1α and HIF2α increased when the shift from high to low oxygen occurred, suggesting that these genes were not expressed before, likely because cells were adapted to the low oxygen tension. When the shift occurs, the cells reacted with an increase of HIF1α and HIF2α. The study of these effects during reprogramming in vitro is critical, and biological repetitions to reinforce this data are underway. Herein, we show that the in vitro reprogramming process may be modulated by environmental oxygen changes.